This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To define the ability of rhesus embryonic stem cells to differentiate into trophblasts. We previously found that showed that the differentiation potential of rhesus embryonic stem cell (ESC)-like lines to form trophoblast cells was variable, with this differentiation potential ranging from nondetectable to consistent. We have evaluated the undifferentiated cells by microarray, and identified significant differences with high biological relevance. Outgrowths in a 2-dimensional culture paradigm from rhesus ESC which give morphological evidence of trophoblast formation also express placental MHC class I surface molecules. In the previous year we have further compared embryonic stem cell differentiation potential. We have now expanded the range of cells available to include embryo-derived rhesus ESC provided by Dr. Shoukhrat Mitalipov at the Oregon National Primate Research Center (ORMES lines), as well as cloned rhesus ESC derived in his lab ((CRES lines). We have expanded these cell lines and conducted preliminary studies using the standard paradigms established in our lab for trophoblast differentiation from human embryonic stem cells: treatment with BMP-4, formation of embryoid bodies and outgrowth culture on 2-dimensional culture surfaces with Matrigel, and embryoid bodies introduced into three-dimensional culture environments, i.e., droplets of solidified Matrigel. Experiments completed thus far indicate that the ORMES cells form expected outgrowths in 2 and 3 dimensions. However, of the two lines evaluated so far (4 others yet to be analyzed), as with some of the rhesus ESC lines from the Thomson lab, there is little detectable epithelial differentiation and thus no indication of trophoblast differentiation. The CRES lines have proven rather difficult to handle, and we will revisit those lines soon. The placenta has the unique property of promoting maternal immune responses that allow recognition of pregnancy, but tolerance of the paternal genetic component of the fetus. The expression of placental MHC class I molecules on rhesus ESC outgrowths from selected lines suggests that these may serve as a possible source of embryonic surrogates to study the immune response to trophoblast exposure in vivo. We have prepared embryoid bodies from rhesus ESC, and have conducted three transfers of embryoid bodies to recipient female rhesus monkeys. The rhesus monkeys were in the stage of the menstrual cycle during the presumed window of implantation, days 4-7 of the luteal phase. One set of embryoid bodies was transferred to the oviduct by laparotomy, and two sets were transferred to different recipients by transcervical transfer, similar to our previous studies with nonsurgical rhesus monkey embryo transfer. In addition, two animals have received sham transcervical transfers: only the transfer pipet was introduced into the uterine cavity and saline vehicle was infused. All animals received progesterone injections to support the receptive endometrium. With tubal transfer, there was no evidence for retention of embryoid body cellular material, however we noted an unexpected induction of a pregnancy-specific macrophage marker, DC-SIGN, in the endometrium in this animal. In addition, although analysis is not completed, one of the two animals that received intrauterine transcervical embryoid body transfers also had DC-SIGN induction. We are working to define whether this is a response to embryoid bodies, the response to placement of the transfer catheter, or simply the induction by prolonged progesterone treatment. This research used WNPRC Stem Cell Resources.